This report describes the modification of polycrystalline gold surfaces with mixed self-assembled monolayers (SAMs) containing COOH and one of the end groups CH3, OH, or NH2, with the aim of modulating the surface physicochemical properties (charge and wettability) and investigating the effect of these factors on the surface amount and secondary structure of fibronectin (FN), chemically attached to these surfaces. It is shown that the SAM composition influenced the substrate surface charge, for all three types of binary mixed SAMs, while only the CH3/COOH SAM was found to influence the substrate surface wettability. It was also found that the covalent binding of FN to the SAMs resulted in alteration in the secondary structure of the protein, in comparison to the structure in its native state. However, there was no straightforward correlation between the SAM composition and the FN surface amount and its secondary structure. Further, a simple method for evaluating the true surface composition of COOH-based mixed SAMs is presented. (C) 2012 Elsevier B.V. All rights reserved.